CN110007032A - A kind of phospholipid detection method and application - Google Patents

Abstract

The invention discloses a phospholipid detection method and application, wherein a phospholipid detection method is eluted with acetonitrile and ammonium acetate; a phospholipid detection eluent, which includes acetonitrile and ammonium acetate, wherein, by volume percentage In total, the acetonitrile is 50% to 95%, and the ammonium acetate is 5% to 50%; the application of a phospholipid detection method is for detecting phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phospholipid Detection of acylethanolamine, glycerophosphatidic acid, sphingomyelin, and lysophosphatidylcholine. The analysis method provided by the invention has the advantages of simple operation, many types of phospholipids to be analyzed, good peak shape, short retention time, convenience, quickness, reliability and practicality.

Description

A kind of phospholipid detection method and application

technical field

The invention belongs to the technical field of phospholipid detection, in particular to a phospholipid detection method and application.

Background technique

Phospholipids are the main components of biological membranes. According to their polar acyl head groups, phospholipids are divided into six categories: phosphatidyl cholines (PC), phosphatidyl ethanolamines (PE), phosphatidyl serines (PS), and phosphatidyl glycerols. Glycerol, PG), glycerophosphatidic acid (PA) and sphingolipid (SM), the Sn-2 acyl chain of phospholipids can be degraded into lysophospholipids, such as lyso-phosphatidylcholine (LPC) ). The phospholipid bilayer is the basic structure of the cell membrane. People's understanding of the life function of phospholipids begins with the basic components of biological membranes. Almost all functions performed by cells at the level of the cell membrane and intracellular membrane are directly or indirectly affected by the localization of phospholipids. And the influence of structural changes, the important functions of phospholipids have been continuously discovered. The composition of membranes in living organisms is not stable, external disturbances and internal changes of living organisms may cause changes in the composition of phospholipids on membranes. The study of the changes of phospholipids on the membrane can help to understand the changes in the metabolic mechanism of cell growth caused by the external environment. Caenorhabditis elegans is an internationally recognized research model organism for phospholipids. Membrane lipids are composed of structural lipids (membrane lipids such as PL) and storage lipids (mainly neutral lipids). The composition of phospholipids is diverse . Changes in polar head groups, glycerol backbone, phospholipids or stereospecificity will strongly affect the biological activity of the organism.

Therefore, rapid quantitative detection of phospholipids is necessary and practical. For the detection of phospholipids, normal-phase or reversed-phase liquid chromatography is generally used at home and abroad. Normal-phase chromatography mostly uses silica gel columns, which generally only respond strongly to phosphatidylcholine and phosphatidylethanolamine, and are sensitive to other types of phospholipids. The desorption ability is weak, and the equilibration time required for the detection is too long, which is not suitable for the rapid analysis of complex biological membrane phospholipids. However, reversed-phase chromatography is based on the difference in hydrophobicity caused by the length of the acyl chain of phospholipid compounds. Therefore, various types of phospholipids are easily eluted in the same time period, resulting in overlapping peak shapes. In addition, there are reports on the detection of phospholipids by mass spectrometry. However, the price is high, the maintenance cost is high, and the ionization of phospholipid molecules is inhibited, which affects the peak shape. In contrast, the high-performance liquid chromatography evaporative light detection method is simple in operation, high in accuracy and low in cost. Some studies have explored related methods, but there are still some problems that need to be improved. Propanol has great damage to the chromatographic column, liquid pump, split ring and other hardware, which will shorten its life, and the reported detection methods can only detect a limited number of phospholipids such as phosphatidylcholine and phosphatidylethanolamine, or the equilibration time Long peaks, tailing peaks, and difficult separation lead to inaccurate quantification. With the increase of phospholipid molecules, the required separation time increases. Selecting appropriate elution gradients and chromatographic parameters is crucial to both separation effect and analysis efficiency.

SUMMARY OF THE INVENTION

The purpose of this section is to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the application to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions may not be used to limit the scope of the invention.

In view of the above-mentioned technical defects, the present invention is proposed.

Therefore, as one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, and provides a phospholipid detection method and application.

In order to solve the above-mentioned technical problems, the present invention provides the following technical scheme: a phospholipid detection method, characterized in that: acetonitrile and ammonium acetate are used for elution.

As a preferred solution of the phospholipid detection method of the present invention, the elution is gradient elution.

As a preferred solution of the phospholipid detection method of the present invention, wherein, the gradient elution is to use the first volume ratio of the acetonitrile and the ammonium acetate to elute at 0 to 2 minutes, and use the second volume of 2 to 20 minutes to elute. The acetonitrile and the ammonium acetate in the volume ratio are rinsed, and then the acetonitrile and the ammonium acetate in the first volume ratio are rinsed;

Wherein, the first volume ratio is 95:5, and the second volume ratio is less than or equal to the first volume ratio.

As a preferred solution of the phospholipid detection method of the present invention, the second volume ratio is one or more of (95:5)～(50:50).

As a preferred solution of the phospholipid detection method of the present invention, the second volume ratio is one or more of 90:10, 80:20, 70:30 or 60:40.

As a preferred solution of the phospholipid detection method of the present invention, wherein, in the elution, the column temperature of the chromatographic column is 30-40°C, and the temperature of the drift tube is 50-55°C.

As a preferred solution of the phospholipid detection method of the present invention, wherein, in the elution, the injection volume is 5-20 μL, the flow rate is 0.8-2.0 mL/min, and the carrier gas pressure is 2.50 bar.

As a preferred solution of the phospholipid detection method of the present invention, the detection interval is 3.125-100 mg/L.

As another aspect of the present invention, in order to solve the above technical problems, a phospholipid detection eluent is provided, which includes acetonitrile and ammonium acetate; wherein, in terms of volume percentage, the acetonitrile is 50% to 95%, so The ammonium acetate is 5% to 50%.

As another aspect of the present invention, in order to solve the above-mentioned technical problems, an application of a phospholipid detection method is provided, which is for detecting phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, glycerophosphatidic acid, Detection of sphingomyelin and lysophosphatidylcholine.

Beneficial effects of the present invention:

The analysis method provided by the invention has the advantages of short retention time, high quality of chromatogram, good separation effect and short time required for separation.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort. in:

Fig. 1 is the UPLC-Q-TOF-MS spectrum of the detection of the phospholipid standard sample in Example 1.

Figure 2 is the detection HPLC-ELSD spectrum of the phospholipid standard mixed sample, wherein Figure 2a is the detection spectrum of Example 2; Figure 2b is the detection spectrum of Example 3; Figure 2c is the detection spectrum of Example 4.

Figure 3 is the HPLC-ELSD spectrum of the phospholipid standard mixed sample at different column temperatures in Example 5, wherein Figure 3a is 30°C, Figure 3b is 35°C, and Figure 3c is 40°C.

Figure 4 is the HPLC-ELSD spectrum of the phospholipid standard mixed sample at different drift tube temperatures in Example 6, wherein Figure 4a is 45°C, Figure 4b is 50°C, and Figure 4c is 55°C.

Figure 5 is the HPLC-ELSD spectrum of the low concentration 0.002 mg/mL phospholipid standard mixed sample of Example 8.

Figure 6 is the HPLC-ELSD spectrum of the high concentration of 0.2 mg/mL phospholipid standard mixed sample in Example 8.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below with reference to specific embodiments.

Many specific details are set forth in the following description to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can do so without departing from the connotation of the present invention. Similar promotion, therefore, the present invention is not limited by the specific embodiments disclosed below.

Second, reference herein to "one embodiment" or "an embodiment" refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of "in one embodiment" in various places in this specification are not all referring to the same embodiment, nor are they separate or selectively mutually exclusive from other embodiments.

The test instruments selected in the embodiment of the present invention are a 2695 type high performance liquid chromatograph (Waters, USA), a 2414 type evaporative light detector, the carrier gas used is nitrogen, and a nitrogen generator is equipped.

Example 1:

Weigh 0.1 mg of phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and glycerophosphatidic acid, respectively, and dissolve them in 1 mL of chloroform-methanol solution (2:1, v/v) to make 100 mg/L. The single standard solution, after passing through a 0.45 μm membrane, was detected by ultra-high performance liquid chromatography time-of-flight mass spectrometry (UPLC-Q-TOF-MS).

Chromatographic experimental conditions are CORTECS UPLC HILIC column (φ2.1mm×150mm, 1.6μm); mobile phase: A is acetonitrile, B is 10mM ammonium acetate aqueous solution (containing 0.1% formic acid, pH≈3.65); elution program: 0 ～2min, 95%A, 5%B; 2～15min, 95%～70%A, 5%～30%B; 15～17min, 70%～60%A, 30%～40%B; 17～17.1 min, 60%～95%A, 40%～5%B; 17.1～20min, 95%A, 5%B; flow rate 0.3mL/min; injection volume: 1μL.

The experimental conditions of mass spectrometry were electrospray positive and negative ion mode (ESI, scanning range m/z 50-1500, scanning time: 0.2s, capillary voltage: 3.5kV, cone voltage: 30V, detector voltage: 1800V, ion source temperature: 100 °C, desolvation gas temperature: 400 °C, collision energy 15V).

This embodiment adopts ultra-high performance liquid phase and uses HILIC column, but because the applicable flow rate of ultra-high performance liquid phase is small and the diameter of the chromatographic column used is relatively small, the elution effect of phospholipids may not be good; Inhibition occurs. The results are shown in Figure 1. Under these conditions, phosphatidylcholine and phosphatidylethanolamine can produce peaks, but they also have the disadvantage of broad peak shape, while phosphatidylserine, phosphatidylglycerol and glycerophosphatidic acid appear ghost peaks or slope peaks. , cannot be integrated quantitatively.

Example 2:

In view of the problem of insufficient method in Example 1, this example adopts a high-performance liquid phase system (HPLC) and a HILIC chromatographic column with a wider diameter for phospholipid analysis.

In order to simplify the sample preparation process, respectively weigh 0.1 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, and mix them in 1 ml of chloroform methanol solution. (2:1, v/v) into a 100 mg/L mixed standard solution, and after passing through a 0.45 μm membrane, it was detected by high performance liquid chromatography. The detection conditions were an XBridge ^TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate was 1.0mL/min, the column temperature was 35°C, the injection volume was 10μL, the drift tube temperature was 50°C, and the gas pressure was 2.50bar.

Elution was performed by mixing acetonitrile with ammonium acetate as the mobile phase. First, take acetonitrile:ammonium acetate=95:5 as the starting point, do 2min isoconcentration elution, slowly reduce the acetonitrile concentration in 15min, reach acetonitrile:ammonium acetate=70:30 at 15min, and reach acetonitrile:ammonium acetate=60:40 at 17min , return to the starting point at 18min, the above ratios are volume ratios. The detection is shown in Figure 2a.

Example 3:

Weigh 0.1 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and mix them in 1 mL of chloroform methanol solution (2:1, v /v) Make up 100mg/L mixed standard solution, pass through 0.45μm membrane, and then enter into high performance liquid chromatography for detection. The detection conditions were an XBridge ^TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate was 1.0mL/min, the column temperature was 35°C, the injection volume was 10μL, the drift tube temperature was 50°C, and the gas pressure was 2.50bar.

Elution was performed by mixing acetonitrile with ammonium acetate as the mobile phase. First start with acetonitrile:ammonium acetate=95:5, do 2min isoconcentration elution, then reduce the acetonitrile concentration, reach acetonitrile:ammonium acetate=90:10 at 8min, reach acetonitrile:ammonium acetate=80:20 at 10min, 17min When acetonitrile:ammonium acetate=70:30, it returns to the starting point after 3min, and the above ratios are all volume ratios. The detection is shown in Figure 2b.

Example 4:

Weigh 0.1 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and mix them in 1 mL of chloroform methanol solution (2:1, v /v) Make up 100mg/L mixed standard solution, pass through 0.45μm membrane, and then enter into high performance liquid chromatography for detection. The detection conditions were an XBridge ^TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate was 1.0mL/min, the column temperature was 35°C, the injection volume was 10μL, the drift tube temperature was 50°C, and the gas pressure was 2.50bar.

Elution was performed by mixing acetonitrile with ammonium acetate as the mobile phase. First start with acetonitrile:ammonium acetate=95:5, do 2min isoconcentration elution, then reduce the acetonitrile concentration, reach acetonitrile:ammonium acetate=90:10 at 3min, reach acetonitrile:ammonium acetate=80:20 at 12min, 15min When acetonitrile: ammonium acetate = 70:30, return to the starting point after 2 minutes, the above ratios are volume ratios. The detection is shown in Figure 2c.

Gradient elution can effectively separate phospholipids of different polarities and shorten the liquid phase analysis time. Isocratic elution cannot effectively separate acidic phospholipids from neutral phospholipids. It is within the scope of the present invention to vary the elution pattern within the scope of the art. The experimental study conducted by the inventor confirmed that the third gradient, namely Example 4, was the best, and seven phospholipids (phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, lysophosphatidyl Choline) peaked within 3 to 15 minutes successively, and the separation effect was good in the mobile phase of 90:10. Slowly reducing the ratio of acetonitrile for 3-12 minutes can effectively separate phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine and phosphatidylethanolamine. A suitable gradient descent rate avoids ghost peaks, maintains good peak shape, avoids baseline elevation, and minimizes overall analysis time.

Example 5:

Weigh 0.1 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and mix them in 1 mL of chloroform methanol solution (2:1, v /v) Make up 100mg/L mixed standard solution, pass through 0.45μm membrane, and then enter into high performance liquid chromatography for detection.

The detection conditions were set as XBridge ^TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate was 1.0mL/min, the injection volume was 10μL, the drift tube temperature was 50°C, and the gas pressure was 2.50bar. Example 4 was selected. The elution mode of , was detected under the conditions that the column temperature was 30°C, 35°C, and 40°C, respectively, and the obtained images were shown in Figures 3a, 3b, and 3c, respectively.

The best peak shape can be obtained at 35°C. If the temperature above 40°C is too high, it is easy to denature the phospholipids, and it is not conducive to the maintenance of the life of the chromatographic column. Select the column temperature of 35℃ as the optimal column temperature parameter.

Example 6:

Weigh 0.1 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and mix them in 1 ml of chloroform methanol solution (2:1, v /v) Make up 100mg/L mixed standard solution, pass through 0.45μm membrane, and then enter into high performance liquid chromatography for detection.

The detection conditions were set as XBridge ^TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate was 1.0mL/min, the column temperature was 35°C, the injection volume was 10μL, and the gas pressure was 2.50bar. In the elution mode, the temperature of the drift tube is 45°C, 50°C, and 55°C for detection, and the obtained spectrum is shown in Figure 4.

Finally, it was found that the peak shape obtained at 50°C was the best, and the elution peak intensity was the highest. The drift tube temperature of 50°C is selected as the drift tube temperature parameter of the present invention.

Example 7:

Weigh 0.02 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and mix them in 1 ml of chloroform methanol solution (2:1, v /v) Make into a 20mg/L mixed standard solution, pass through a 0.45μm membrane, and then enter into high performance liquid chromatography for detection. Three consecutive injections, manual integration, and calculation of retention time precision and peak area precision.

Weigh 0.01 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and mix them in 1 ml of chloroform methanol solution (2:1, v /v) Make up 10mg/L mixed standard solution. Add the mixed standard to the sample and prepare a blank sample without the mixed standard. After passing the sample through a 0.45 μm membrane, it was detected by high performance liquid chromatography.

The detection conditions were set as XBridge ^™ HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate was 1.0mL/min, the column temperature was 35°C, and the injection volume was 10μL, and the elution mode of Example 4 was selected. The drift tube temperature was 50°C and the gas pressure was 2.50 bar.

As shown in Table 1 and Table 2, the obtained standard recovery rate is 87.24-114.71%, the retention time precision is 0.03-0.72%, and the peak area precision is 2.07-8.77%. The index shows that the detection method adopted in this example has good precision and the recovery rate is within an appropriate range, which proves that the detection method is good.

Table 1 The present embodiment detects retention time and peak area precision of phospholipids

Table 2 The recovery rate of standard addition for the detection of phospholipids in the present embodiment

Example 8:

Weigh 1 mg of phosphatidylglycerol, glycerophosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, respectively, and dissolve them in 1 mL of chloroform methanol solution (2:1, v/v ) into a 1mg/mL mixed standard solution, and gradually diluted to 0.5mg/mL, 0.2mg/mL, 0.1mg/mL, 0.05mg/mL, 0.025mg/mL, 0.0125mg/mL, 0.00625mg/mL, 0.003125 mg/mL, 0.002mg/mL and 0.001mg/mL, after passing through a 0.45μm membrane, were detected by high performance liquid chromatography.

Experimental studies have confirmed that the sample concentration is too low, the signal intensity is too poor, and the noise is large, as shown in Figure 5; the concentration is too high, and the flat-top peak is prone to appear, as shown in Figure 6; the sample concentration is selected at the concentration of 3.125 ~ 100mg/L interval, suitable for sample detection.

The detection conditions are set as follows: the chromatographic column is an XBridge ^TM HILIC column (φ4.6mm×250mm, 5μm), the flow rate is 1.0mL/min, the injection volume is 10μL, the column temperature is 35°C, the drift tube temperature is 50°C, and the gas The pressure was 2.50 bar, and the elution mode of Example 4 was selected for detection, and the results were shown in Table 3.

The linear range and detection limit of table 3 the present invention detects phospholipid

Example 9:

In this study, inactivated Escherichia coli (strain OP50) was used as food source, and N ₂ (wild-type) C. elegans was cultivated by solid culture method.

Accurately weigh 1 mL of frozen C. elegans into a stoppered test tube, add 1.2 mL of deionized water, 1.5 mL of chloroform, 3 mL of methanol, vortex, mix well, and sonicate for 30 minutes; add 1.5 mL of chloroform, vortex, mix well, and sonicate 30min; add 1.5mL of water, vortex, mix well, sonicate for 10min with a cell disruptor; centrifuge at 5000rpm for 10min. After centrifugation, the solution was divided into three layers, the upper layer was methanol and water layer, the thin white layer in the middle was bacterial residue, the lower layer was chloroform layer, and the phospholipids existed in the chloroform layer. Transfer the lower layer solution to a glass bottle; add 1.5 mL of chloroform to the remaining solution and residue, vortex, mix well, sonicate for 30 min and then centrifuge (4500 rpm, 10 min); mix the lower clarified solution with the previous solution, blow with nitrogen, The obtained solid was dissolved in 1 mL of chloroform/methanol solution (2:1, v/v), and stored at -20°C until testing.

After filtration through a 0.45 μm filter membrane, the phospholipid composition was determined under the detection conditions set in Example 2. The measurement results are shown in Table 4.

Table 4 Detection of Caenorhabditis elegans phospholipid composition and content

It can be seen from the data results obtained in the examples that the standard deviation is between 0.1 and 0.8, and the separation efficiency is good; among the seven phospholipids, the content of phosphatidylcholine is the highest, followed by the higher content of phosphatidylethanolamine, sphingomyelin and glycerophosphatidic acid.

The invention uses acetonitrile and ammonium acetate as mobile phases, can perform qualitative and quantitative detection of complex phospholipid samples, has better detection accuracy, and does not cause chromatographic column fillers compared with mobile phases containing n-hexane and isopropanol. The damage will accelerate the aging of the chromatographic column, and it will not cause damage to the detection hardware and prolong the service life of the detection instrument.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent substitutions without departing from the spirit and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims (10)

1. a kind of phosphatide detection method, it is characterised in that:

It is eluted with acetonitrile and ammonium acetate.

2. phosphatide detection method as described in claim 1, it is characterised in that: the elution is gradient elution.

3. phosphatide detection method as claimed in claim 2, it is characterised in that: the gradient elution, with first when being 0~2min The acetonitrile of volume ratio and ammonium acetate elution, with the acetonitrile and the ammonium acetate of the second volume ratio when 2~20min Elution reuses the acetonitrile and the ammonium acetate elution of first volume ratio；

Wherein, first volume ratio is 95:5, and second volume ratio is less than or equal to first volume ratio.

4. phosphatide detection method as claimed in claim 3, it is characterised in that: second volume ratio is (95:5)~(50:50) It is one or more.

5. phosphatide detection method as claimed in claim 3, it is characterised in that: second volume ratio is 90:10,80:20,70: 30 or 60:40's is one or more.

6. the phosphatide detection method as described in Claims 1 to 5 is any, it is characterised in that: the column temperature of the elution, chromatographic column is 30~40 DEG C, drift tube temperature is 50~55 DEG C.

7. the phosphatide detection method as described in Claims 1 to 5 is any, it is characterised in that: the elution, sample volume are 5~20 μ L, flow velocity are 0.8~2.0mL/min, nebulizer gas pressure 2.50bar.

8. the phosphatide detection method as described in claim 1,2, it is characterised in that: detection interval is 3.125~100mg/L.

9. a kind of phosphatide detects eluent, it is characterised in that: including,

Acetonitrile, ammonium acetate；

Wherein, by volume percent, the acetonitrile is 50%~95%, and the ammonium acetate is 5%~50%.

10. a kind of application of phosphatide detection method, it is characterised in that: for detecting phosphatidyl choline, phosphatidyl glycerol, phosphatidyl The detection of serine, phosphatidyl-ethanolamine, glycerophosphatide acid, sphingomyelins, lysophosphatidyl choline.